Analysis of Favipiravir in Human Plasma

Significance of the Topic

Favipiravir, an RNA polymerase inhibitor initially developed against influenza, has become an important candidate for antiviral research due to its broad-spectrum activity. Reliable quantification of favipiravir in biological matrices such as human plasma is critical for pharmacokinetic studies, therapeutic monitoring and drug development.

Objectives and Study Overview

The primary aim of this study was to develop and validate a robust, sensitive LC-MS/MS method for the quantification of favipiravir in human plasma. Key goals included establishing linearity across a wide concentration range, evaluating precision and accuracy, and confirming long-term stability and repeatability of the analytical system.

Instrumentation

This analysis employed an ultra-high-performance liquid chromatography system (Nexera™ X2 UHPLC) coupled to a triple quadrupole mass spectrometer (Shimadzu LCMS-8060) with heated electrospray ionization (ESI). The chromatographic separation utilized a Shim-pack Scepter C18-120 column (50 mm × 2.1 mm I.D., 1.9 µm) under a gradient of 0.05% formic acid in water and 0.05% formic acid in acetonitrile. The mass spectrometer was operated in positive multiple reaction monitoring (MRM) mode monitoring transitions m/z 157.7→85.1 and 157.7→113.2 for favipiravir and m/z 159.7→85.1 and 159.7→113.2 for the [13C,15N]-labelled internal standard.

Methodology and Sample Preparation

Human plasma samples were spiked with favipiravir and a stable isotope internal standard. Protein precipitation was performed by adding isopropyl alcohol, internal standard solution and acetonitrile, followed by shaking and centrifugation. The clear supernatant was directly injected (1.0 µL) into the LC-MS/MS system.

Main Results and Discussion

A seven-point calibration curve covering 1–100 µg/mL demonstrated excellent linearity (R2 = 0.9998). Intra-day precision ranged from 1.7% to 6.5% RSD, and accuracy was 96–103%. Quality control samples at low (3 µg/mL), medium (50 µg/mL) and high (90 µg/mL) levels showed repeatability RSD of 1.7–4.5% and accuracy of 98–104%. Between-day reproducibility over three days yielded RSD values of 0.1–5.2% and accuracy of 89–102%. Robustness was confirmed by 100 consecutive injections at 10 µg/mL, resulting in RSDs of 2.9% for favipiravir and 3.2% for the internal standard, indicating stable performance and consistent sensitivity.

Benefits and Practical Applications

This validated LC-MS/MS method offers high selectivity, rapid analysis time and minimal sample preparation, making it well suited for pharmacokinetic studies, therapeutic drug monitoring and high-throughput screening in clinical and research laboratories.

Future Trends and Applications

Advances may include integration with automated sample preparation platforms, application to other biological matrices and adaptation for simultaneous quantification of multiple antiviral agents. Enhanced high-resolution MS could further improve sensitivity and selectivity for emerging drug candidates.

Conclusion

The developed LC-MS/MS assay for favipiravir demonstrates excellent linearity, precision, accuracy and long-term robustness, supporting its use in routine bioanalysis and drug development workflows.

Reference

• No specific literature references were provided in the original application note.